Inactivation of Fish Pathogens with Ozone

Ozone Reaction and Inactivation of Fish Pathogens

(To obtain more information on recirculating aquaculture systems, a complete set of tables, figures and references shown on this and related pages please refer
to the book "Recirculating Aquaculture Systems, 2nd Edition", M. B. Timmons, et al, 2002, Cayuga Aqua Ventures, Ithaca, NY. You can obtain a copy of the
publication from Cayuga Aqua Ventures by visiting their website at www.c-a-v.net.)

Ozone oxidation can kill microorganisms, but requires maintaining a certain dissolved ozone concentration in the water
for a given contact time. Disinfecting efficiency depends upon the product of the ozone residual concentration and its
contact time. An ozone contact vessel provides the time necessary for the ozone residual to react with and inactivate
pathogenic microorganisms. Disinfecting waters may require maintaining a residual ozone concentration of 0.1–2.0
mg/L in a plug-flow type contact vessel for periods of 1–30 min, depending upon the target microorganism (Wedemeyer,
1996). In commercial aquaculture applications, it is extremely difficult to maintain residual concentrations above 1 mg/L;
above 2 mg/L is almost impossible with conventionally available equipment.

Ozone exposure experiments with bacterial cells have indicated changes in membrane structure which lead to leakage of
protein and nucleic acid, and also lipid oxidation, while the intracellular components, protein, and DNA, remain intact
(Komanapalli and Lau, 1996). By prolonged ozone exposure, cell viability is reduced with a more significant increase in
lipid oxidation and protein and nucleic acid leakage.

In general, bacteria and viruses pathogenic to salmonids are highly sensitive to residual ozone in water (see Tables 12.1
and 12.4). Dose-response estimates of this sensitivity are fairly precise in demand-free water (inorganic buffers,
distilled water), indicating 99.9% inactivation or more in the 0.01–0.10 mg/L residual concentration range. In batch
experiments with natural water exerting an ozone demand, residual concentration tends to drop rapidly, making reliable
dose-response estimates more complicated. As a rule, higher residual concentrations, i.e., 0.1–0.2 mg/L in natural sea-
, brackish-, and freshwater, and 0.3–0.4 mg/L in fish farm effluents, seem necessary to obtain the legally required
inactivation level.

In dose-response trials, two-stage logarithmic inactivation curves have been obtained (Liltved et al. 1995; Liltved and
Landfald, 1995). These were characterized by a rapid inactivation initially, followed by a decreasing inactivation rate
with exposure time. Such kinetics could be explained by reduced ozone concentration during the course of exposure,
and have also been experienced by other investigators, even in ozone demand-free water (Katzenelson et al. 1974;
Colberg and Lingg, 1978; Vaughn et al. 1987). In practical ozonation of fish farm influent and effluent water, it is
important that the ozone dose is high enough to account for the initial demand, thereby establishing a sufficient residual
concentration for the required contact time.

In demand-free water, dissipation of ozone will still be observed due to the demand exerted by the added
microorganisms. This demand will depend on type of organism, the preparation, and washing of the inoculum prior to
supplementation, and the density of organisms in the ozonated suspension. In natural waters and in waters found within
recirculating systems, additional ozone will be lost in reactions with organics and other compounds. According to ozone
demand tests on a high quality trout stream water that is being ozone disinfected at the US Fish and Wildlife Service
Lamar National Fish Hatchery (Lamar, Pennsylvania), an ozone concentration of 2–4 mg/L must be transferred to
maintain a 0.2 mg/L ozone residual concentration after 10 minutes (Steven Summerfelt, unpublished data). Cryer
(1992) reported similar ozone demand results in tests on surface water supplies that were being disinfected at US Fish
and Wildlife Service salmonid hatcheries in North America. All of the surface water supplies examined in these studies
exhibit relatively high water quality with low concentrations of oxidizable organic material, iron, and manganese (Cryer,
1992; Steven Summerfelt, unpublished data), yet the ozone demand created still reduced the half-life of ozone to less
than a few minutes. In comparison, the half-life of ozone dissolved in pure water at 20ºC is 165 minutes (Rice et al.
1981).

The ozone demand of water within RAS, which contains much higher levels of organic material and nitrite, creates a
short ozone half-life, e.g., less 15 seconds, and makes maintaining an ozone residual difficult (Bullock et al. 1997). For
this reason, it is difficult to add enough ozone to achieve microbial inactivation in recirculating systems. In recirculation
systems ozone is most often applied at doses that promote water quality improvement (Brazil, 1996; Bullock et al. 1997;
Summerfelt and Hochheimer 1997; Summerfelt et al. 1997). Using ozone in recirculating systems can reduce fish
disease simply by improving water quality, which reduces or eliminates environmental
sources of stress (Bullock et al. 1997). These studies, as well as experience with ozone application at numerous
commercial recirculating systems, indicates that both water quality and fish health can be improved by adding
approximately 13–24 g ozone for every 1.0 kilogram of feed fed to a recirculating system.

Ozonation may enhance fine solids removal by changing particle size rather than separating particles from water. As an
unstable reactive gas, ozone splits large organics into smaller biodegradable materials that can be more easily removed
by heterotrophic bacteria. Conversely, ozone can polymerize metastable organics leading to enmeshment, direct
precipitation, bridging, or adsorption (Reckhow et al. 1986). Ozone has been used sometimes with mixed success in a
variety of aquaculture systems to remove color and turbidity (Colberg and Lingg, 1978; Williams et al. 1982; Paller and
Lewis, 1988; Brazil, 1996; Summerfelt et al. 1997). Effects of ozonation upon particle size change in recirculating
systems are still not clearly defined. The function of ozone is complicated; both qualitative and quantitative impacts of
ozonation may be specific to a given system (Grasso and Weber, 1988). There are also concerns that even low ozone
residuals may cause gill adhesions and mortality in fish exposed to freshly ozonated water (Rosenlund, 1975).

Besides being a method for water improvement in recirculation systems, ozone is valued for its high virucidal activity,
Table 12.1. (Click here to see the Table)

Among fish pathogenic viruses, high sensitivity toward ozone has generally been reported. This also applies to viruses
with high UV resistance, i.e., IPNV and WSBV (Liltved et al. 1995; Chang et al. 1998). As viral diseases have become a
major threat in worldwide aquaculture, the virucidal properties of ozone will certainly be more valued in the future, both in
intake and effluent water disinfection. Ozone-treated water has also proven useful for washing fertilized eggs (Arimoto
et al. 1996) and for reducing or eliminating potential pathogens associated with live prey such as rotifers in marine larval
production systems (Davis and Arnold, 1997; Theisen et al. 1998).